Recently, analytical elements such as a macro-array sheet and a micro-array sheet are widely utilized in gene analysis technology in the biological and medical fields. These analytical elements are used to analyze nucleic acids such as DNA and RNA, their fragments, and their duplicates such as PCR products. The analysis is performed by detecting the nucleic acids utilizing hybridization, which is one of biochemically specific binding reaction. The former macro-array sheet is made of porous sheet of polyamide resin or the like. For the analysis using the macro-array sheet, a large number of nucleic acid fragments (probe molecules) such as DNA fragments are first fixed to the porous sheet by entanglement. The binding reaction is carried out using (target) sample nucleic acid fragments labeled with radioisotope (RI). The latter micro-array is composed of solid carrier such as surface-treated slide glass plate. For the analysis using the micro-array sheet, the probe molecules are first fixed to the surface of the solid carrier. The binding reaction is carried out between the probe molecules and sample nucleic acid fragments labeled with fluorescent compound.
However, the names of macro-array sheet and micro-array sheet are not always employed distinctly to mean each of the above-described analytical elements.
The gene analysis utilizing a macro-array sheet is advantageous because it can be carried out using the conventional autoradiography.
The conventional procedures for detecting a nucleic acid such as DNA are generally conducted by the process comprising the following steps:
(1) preparing a number of single-stranded nucleic acid fragments (probe molecules: generally employed are those of which base sequences are already known); spotting in series an aqueous solution containing the fragments on a macro-array sheet using a spotter so that a large number of spots can be densely placed on the sheet to form a matrix composed of pores to which probe molecules are fixed by entanglement, whereby a large number of probe molecule spots in the form of dots are produced;
(2) bringing single-stranded sample nucleic acid fragments (to be analyzed) with a radioisotope label (RI, for instance, 32P and 33P) contained in an aqueous solution into contact with the macro-array sheet (for instance, by immersing the macro-array sheet in an aqueous solution of the radioisotope-labeled sample nucleic acid fragments placed in a specific vessel) to fix target nucleic acid fragments to the macro-array sheet by hybridization with the probe molecules; namely, the target complementary nucleic acid fragments in the sample nucleic acid fragments are bound to the probe molecules in the spot by hybridization;
(3) removing unfixed radioisotope-labeled sample nucleic acid fragments from the macro-array sheet by washing;
(4) drying and placing the macro-array sheet on a radiographic film for detecting radiation coming from the radioisotope-labeled target nucleic acid fragments by autoradiography, whereby the binding information of the fixed target nucleic acid fragments (for instance, presence and amount of fixed fragments) are obtained; and
(5) determining at least local base sequence information of the target nucleic acid fragments according to complementation principle, in the case that the base sequence of the probe molecules is previously known.
Thus, a large number of genes are simultaneously analyzed in their expression, mutation, and polymorphism, utilizing the above-described technology.
Recently, a radiation image recording and reproducing method utilizing a radiation image storage panel (which is also named “imaging plate” or “stimulable phosphor sheet”) has been developed for performing autoradiography of radioisotope-labeled biological specimen and polymers originating from living body, in place of the conventional autoradiography using a radiographic film.
The radiation image recording and reproducing method utilizes stimulable phosphor (i.e., radiation image storage phosphor) which absorbs and stores radiation energy when it is exposed to radiation such as X rays, and thereafter produces emission in an amount proportional to the stored radiation energy when it is irradiated with electromagnetic wave (stimulating light) such as visible light or infrared rays, and is generally carried out by a procedure of the following steps:
applying to a stimulable phosphor sheet containing stimulable phosphor a radiation transmitted through or emitted by a target subject, whereby recording the radiation image in the phosphor sheet;
scanning sequentially the phosphor sheet with a stimulating light such as laser light, whereby the phosphor sheet sequentially produces stimulated emission;
photoelectrically detecting the stimulated emission to obtain a series of electric image signals (digital signals); and
storing in an appropriate recording means the digital signals as such or after being subjected to various image processings for forming a visible image.
The autoradiography according to the above-described radiation image recording and reproducing method which utilizes a stimulable phosphor sheet is considered to be important autoradiogaphic technology, because it has various-advantageous features, for instance, it gives a radiation image with high sensitivity even if the amount of radiation coming from the radioisotope-labeled specimen is extremely small, and it gives an image information of digital data which is easily subjected to various image processing procedures and is easily stored.
The autoradiographic procedure utilizing the stimulable phosphor sheet for measuring radiation coming from radioisotope-labeled target molecules is already reported. For instance, Human Molecular Genetics, 1999, Vol. 8, No. 9, 1715–1722 describes that a target molecule can be detected by the steps of producing a large number of spots of DNA fragments (probe molecules) on a porous sheet, hybridizing radioisotope-labeled sample DNA fragments complementary to the probe molecules on the porous sheet, and carrying out the autoradiographic process by placing the porous sheet on a stimulable phosphor sheet.
The gene analysis utilizing a porous sheet such as a macro-array sheet enables to detect radioisotope-labeled target molecules with a high sensitivity when an autoradiographic process is performed utilizing the stimulable phosphor sheet. It has been noted by the inventor, however, that the spots of probe molecules are apt to spread on the porous sheet when a probe molecule solution is spotted on the porous sheet. Accordingly, it is difficult to satisfactorily increase a density of spots (i.e., number of spots per unit area) produced on the porous sheet. Moreover, if the area of spot of probe molecules spreads on the porous sheet, the area in which the radioisotope-labeled target molecules hybridized with the probe molecules also increases. In the increased spot area, the density of the hybridized target molecules decreases. Accordingly, the autoradiography produces a spot image at a less sensitivity, with large noise.
The noise of information is also produced by the fact that the target molecules are attached to the porous sheet not by hybridization.